Compositions comprising PEDF and uses of same in the treatment and prevention of ovary-related syndromes

ABSTRACT

A method of treating or preventing an ovary-related syndrome associated with infertility in a subject in need thereof is provided. The method comprising administering to the subject a pharmaceutical composition comprising an active ingredient consisting of pigment epithelium-derived factor (PEDF) and a pharmaceutically acceptable carrier, thereby treating or preventing the ovary-related syndrome associated with infertility in the subject.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/509,622 filed on Nov. 15, 2012, which is a National Phase of PCTPatent Application No. PCT/IL2010/000936 having International filingdate of Nov. 11, 2010, which claims the benefit of priority under 35 USC119(e) of U.S. Provisional Patent Application No. 61/260,415 filed Nov.12, 2009, the contents of which are incorporated herein by reference intheir entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 60885SequenceListing.txt, created on Feb. 16,2015, comprising 35,328 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates tocompositions comprising PEDF and uses of same in the treatment andprevention of ovary-related syndromes.

Unlike any other organ, the female reproductive organs (i.e., ovary,uterus, and placenta) exhibit cyclic regulation of angiogenesis [1]. Inthe ovary, regulation of angiogenesis is critical for achieving ahealthy mature oocyte. During folliculogenesis, the primordial andprimary follicles are avascular and receive nutrients and oxygen bypassive diffusion from stromal blood vessels. The vascular sheath thatdevelops around each follicle is confined to the theca cells layer,whereas the granulosa cells remain avascular until ovulation, isolatedfrom direct blood supply by the “blood follicular barrier”. Thus, thematuring follicle remains avascular before ovulation, implying thatregulatory mechanism must be present to prevent premature follicularvascularization.

Studies showed that culture media, conditioned by theca cells, stimulateproliferation and migration of endothelial cells regardless of thedevelopmental stage of the follicle. However, Granulosa cells from earlyfollicular phase inhibited migration and proliferation of endothelialcells, while towards ovulation, prior to becoming part of the highlyvascular corpus luteum (CL) [3], they stimulated migration andproliferation of endothelial cells [4].

To date, extensive research has been performed to characterizepro-angiogenic factors in the ovary. Indeed, vascular endothelial growthfactor (VEGF) and fibroblast growth factor 2 (FGF2) were shown to playan important role in the regulation of ovarian angiogenesis. However,only very few studies were conducted to find follicularanti-angiogenesis factors that prevent vessels penetration duringfolliculogenesis [5].

Ovarian stimulation is used with the intention of retrieving a highnumber of oocytes in order to improve the outcome of assistedreproductive treatments. However, administration of high doses ofexogenous gonadotropins may lead to ovarian hyperstimulation syndrome(OHSS). This syndrome appears to be induced by the ovarian release ofvasoactive-angiogenic substances which results in vascularhyperpermeability, leakage and shift of fluids from blood vessels intothe extravascular space leading to consequent clinical manifestationsincluding ascites and edema. Since severe OHSS is potentiallylife-threatening, that can occur in an otherwise healthy young womenundergoing fertility treatments, much effort is made to prevent thisiatrogenic complication. VEGF was recently pointed out as a crucialprotein participating in the development of OHSS (reviewed in [6]) andthe inhibition of the VEGF system could prevent OHSS from occurring. Forexample, Bevacizumab (Avastin™), a humanized monoclonal antibody thatrecognizes and blocks vascular endothelial growth factor A was suggestedas the premium treatment [7]. However, serious adverse have beenreported such as bowel perforation, heart attack and stroke.

In a similar manner to the ovarian follicle, the endometrium alsoundergoes cyclical changes in the course of the ovulatory cycle.Angiogenesis is reinitiated in the uterus during the follicular phaseand continues through the luteal phase of the menstrual cycle due tohormonal induction[1]. In both in vivo and in vitro human models, it hasbeen demonstrated that endometrial angiogenesis is regulated by VEGF,and its expression is significantly increased by estradiol (E2) andprogesterone (P) [8].

Endometriosis is a pathological condition characterized by ectopicendometrial implants, commonly in the peritoneal cavity. Activeendometriosis is characterized by hypervascularization both within andsurrounding the implant. A higher VEGF level has been observed in theperitoneal fluid of patients with endometriosis and its production isstimulated by both E2 and P [8].

Pigment epithelium-derived factor (PEDF) is a non-inhibitory member ofthe serine protease inhibitors (serpin) superfamily, which was firstdescribed as a neurotrophic factor, able to promote and support thegrowth of neuronal cells [9]. However, it was later shown that besidesits neurotrophic function, PEDF is also a potent, natural inhibitor ofangiogenesis [10]. Importantly, its anti-angiogenic activity is fargreater than that of any other known endogenous factor. Theanti-angiogenic effect of PEDF has been extensively investigated in theeye, demonstrating its role in decreasing abnormal neovascularization,mainly by inhibiting the stimulatory activity of several strongpro-angiogenic factors, such as VEGF [11]. Although originallydiscovered in culture media of retinal pigment epithelial cells, PEDF iswidely expressed throughout the body: the nervous system, ovary,uterine, liver [12] and plasma [13]. Despite the significant expressionof PEDF in the reproductive system, there is only limited data about itsfunction in the ovary [14] and uterus [15, 16] referring mainly toregulation in cancer.

U.S. Patent Application Number 20080274967 relates to the use ofphosphorylated PEDF for the treatment of ovarian cancer.

U.S. Patent Application Numbers 20030216286 and 20040014664 teaches theuse of PEDF for inhibiting ovarian and endometrial neovascularizationsuch as for use as a contraceptive by attenuating neovascularizationassociated with ovulation, implantation of an embryo and placentaformation.

International Patent Publication Number WO2007033215 teachesanti-angiogenic compositions for the treatment of endometriosis andovarian hyperstimulation.

U.S. 20040161423 teaches polymer modified PEDF for the treatment ofendometriosis.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of treating or preventing an ovary-relatedsyndrome associated with infertility in a subject in need thereof, themethod comprising administering to the subject a pharmaceuticalcomposition comprising an active ingredient consisting of pigmentepithelium-derived factor (PEDF) and a pharmaceutically acceptablecarrier, thereby treating or preventing the ovary-related syndromeassociated with infertility in the subject.

According to some embodiments of the invention, the ovary-relatedsyndrome associated with infertility is selected from the groupconsisting of ovarian hyperstimulation, endometriosis, infertility andpolycystic ovarian syndrome

According to some embodiments of the invention, the subject is treatedwith an ovarian stimulating medication.

According to some embodiments of the invention, the ovarian stimulatingmedication comprises gonadotropin stimulation.

According to some embodiments of the invention, when the ovary-relatedsyndrome associated with infertility is ovarian hyperstimulation, theadministering is effected at the acute phase of the hyperstimulation.

According to some embodiments of the invention, when the ovary-relatedsyndrome associated with infertility is ovarian hyperstimulation theadministering is effected concomitant with the ovarian stimulatingmedication administration.

According to some embodiments of the invention, when the ovary-relatedsyndrome associated with infertility is polycystic ovarian syndrome, theadministering is effected prior to and/or concomitant with thegonadotropin stimulation at the follicular phase.

According to some embodiments of the invention, when the ovary-relatedsyndrome associated with infertility is endometriosis, the administeringis chronic.

According to some embodiments of the invention, administering iseffected at a dosage range of 0.02-0.4 mg/kg.

According to some embodiments of the invention, the administering iseffected at a dosage range of 0.162-0.32 mg/kg.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising an activeingredient consisting of PEDF for use in the treatment or prevention ofan ovary-related syndrome associated with infertility.

According to an aspect of some embodiments of the present inventionthere is provided a unit dosage form comprising 1-25 mg PEDF.

According to an aspect of some embodiments of the present inventionthere is provided a unit dosage form comprising 9-20 mg PEDF.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-D are images showing the expression of PEDF in the ovary. FIG.1A—Histological sections of mouse ovaries labeled with anti PEDFantibody and Hoechst as a nuclear marker. PEDF is observed within thefollicle in the oocyte and the granulosa cells (arrow). Lower paneldescribes higher magnification of the follicle. FIG. 1B—Freshlyisolated, ovarian GV (germinal vesicle) oocytes (top, synchronized micetreated with 5 IU PMSG) and ovulated MII oocytes (bottom; from PMSG (5IU) and hCG (7 IU) superovulated mice) labeled with anti PEDF antibody.PEDF is produced only by granulosa cells FIGS. 1C-D—Autoradiograph of arepresentative PCR analysis (X35 cycles) demonstrating expression ofPEDF mRNA (P). FIG. 1C—primary granulosa cells obtained from folliclesof 27 days old ICR mice after 3 days of estrogen administration andcultured for additional 7 days before mRNA extraction. Control (GAPDHprimers—G). Marker (M). FIG. 1D—Extraction of mRNA from batches of 100oocytes, demonstrating lack of PEDF mRNA (P) expression in ovarian GVoocytes and ovulated MII oocytes. Control (actin primers—A); marker (M).

FIGS. 2A-B are images showing that PEDF is expressed and secreted bygranulosa cells. Primary granulosa cells were cultured for 7 days toreach hormonal quiescence; Western blot analysis was effected with aspecific PEDF antibody; cell lysates were calibrated with Actin antibody(Two upper panels) and TCA precipitation (Lower panel). FIG. 2A—Primarygranulosa cells obtained from follicles of 27 days old ICR mice after 3days of estrogen administration. FIG. 2B—Human primary granulosa cells(Helsinki 167/09) harvested from follicles of women undergoing IVFtreatment.

FIGS. 3A-E show hormonal regulation of PEDF and VEGF in an opposingmanner. PEDF and VEGF expression in primary granulosa cells before andafter ovulation is shown in FIGS. 3A-B. Granulosa cells were isolatedfrom follicles of PMSG (5 IU) primed, 7 weeks old ICR mice (Gr) or fromoviductal ampullae of superovulated 7 weeks old ICR mice (5 IU PMSG; 7IU hCG); (Gr+). FIG. 3A—Autoradiograph of a representativesemi-quantitative PCR analysis (X35 cycles) of primary mouse granulosacells of PEDF (P) and VEGF (V) mRNAs occurring after in vitro hCGstimulation. Control (GAPDH primers—G). FIG. 3B—RNA extracted fromprimary granulosa cells that were subjected to qPCR analysis withspecific primers for PEDF and VEGF and calibrated with the endogenouscontrol HPRT1. Bars represent Mean±SEM, 6 mice/treatment,(*)—significantly different from control value (P<0.05; T test). Theeffect of hCG on primary human granulosa cells is shown in FIG. 3C—Humanprimary granulosa cells (Helsinki 167/09) were harvested from folliclesof women undergoing IVF treatment. IVF protocol includes stimulationwith hCG to induce ovulation and oocyte retrieval 36 hrs later. Theseprimary granulosa cells were cultured for additional 5-10 days post hCGadministration. TCA precipitation was performed from culture media oneach day between days 5-10. PEDF proteins were detected using WBanalysis with specific antibody. FIGS. 3D-E show that Estrogen andProgesterone downregulate PEDF. TCA precipitation of conditioned mediaof human primary granulosa cells that were pre-cultured for 7 days toreach hormonal quiescence and treated with increasing amount of Estrogen(E2) and Progesterone (P). PEDF proteins were detected using Westernblot analysis with a specific antibody.

FIGS. 4A-D are graphs showing that induction of OHSS reduces PEDFlevels. Excess gonadotropins alters the production of ovarian PEDF andVEGF (FIGS. 4A-B). Ovarian hyperstimulation syndrome (OHSS) was inducedin 5 weeks ICR female mice by 3 consecutive daily injections of PMSG (20IU) followed 24 hours later by hCG (7 IU) stimulation (OHSS). Thecontrol group was administrated with PMSG (5 IU), followed by hCG (7 IU)48 hours later (control). Animals were scarified 48 hr after hCGadministration, ovaries excised and ovarian mRNA was extracted. qPCRanalysis with specific primers for VEGF (FIG. 4A) and PEDF (FIG. 4B) wasperformed and calibrated by the endogenous control HPRT1. Bars representMean±SEM, 10 mice/treatment, (*)—significantly different from controlvalue (P<0.05; T test). Changes in the balance between PEDF and VEGF ingranulosa cells are shown in FIGS. 4C-D. Granulosa cells were obtainedfrom 5 weeks ICR mice as follow: (I) Single treatment of PMSG (5 IU;PMSG1); (II) administration of PMSG (20 IU) for sequential 3 days(PMSG3); (III) administrating of hCG (7 IU) after signal treatment ofPMSG (5 IU; PMSG1+hCG), and (IV) 3 sequential days of PMSG (20 IU)followed by hCG (7 IU; PMSG3+hCG). Subsequently, cells were subjected toRNA extraction and qPCR analysis using specific primers for VEGF andPEDF and calibrated by endogenous control HPRT1. Bars representMean±SEM, 8 mice/group, (*)—significantly different from control value(P<0.05; Ttest and Mann-Whitney).

FIGS. 5A-D show that PEDF can be used as a treatment for OHSS.

(I) ovarian hyperstimulation syndrome (OHSS) was induced in female miceby 3 consecutive daily injections of PMSG (20 IU) followed 24 hourslater by hCG (7 IU) (OHSS) along with intravenously injections of PBS onday 1 and 3 of PMSG stimulation. (II) OHSS+PEDF: Mice were injected withPEDF (2 mg/kg/day) on days 1 and 3 of PMSG (20 IU) followed 24 hourslater by hCG (7 IU). (III) Control group was administrated with PMSG (5IU), followed by hCG (7 IU) 48 hours later. PEDF prevents edema andascites (FIGS. 5A-B). FIG. 5A—Development of edema was assessed by theincrease in body weight (Δgr) throughout the treatments. FIG. 5B—Evansblue was injected intravenously 48 hours after hCG administration andperitoneal lavage was performed to collect fluids that leaked into theabdomen. Dye concentration in the fluids was analyzedspectrophotometrically. Bars represent Mean±SEM, 16 mice/treatment.(*)—significantly different from control value (P<0.05; two way ANOVA).PEDF inhibits OHSS angiogenesis by regulation of VEGF (FIGS. 5C-D). FIG.5C—Histological sections of mice ovaries (Control, OHSS and OHSS+PEDF)labeled with CD34 antibody and Hoechst as a nuclear marker. FIG. 5D—qPCRanalysis with specific primers against VEGF, calibrated by theendogenous control HPRT1. Bars represent Mean±SEM, 10 mice/treatment,(*)—significantly different from control value (P<0.05; two way ANOVA).

FIGS. 6A-C show PEDF expression in the endometrium during the mouseestral cycle. FIG. 6A—Cartoon illustration of the estrous cycle in themouse. FIGS. 6B-C—Seven weeks ICR female mice were synchronized bypre-stimulation for superovulation PMSG (5 IU for 48 hr) followed by HCG(7 IU for additional 48 hours). Endometrium samples were collected fromday diestrus 2 till disetrus 1 of the next cycle. On each day, 2 micewere scarified and the endometrium layer was subjected to qPCR analysiswith Specific primers against PEDF (FIG. 6B) and VEGF (FIG. 6C).Calibration was done with endogenous control HPRT1.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates tocompositions comprising PEDF and uses of same in the treatment andprevention of ovary-related syndromes.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

PEDF is an anti-angiogenic factor which is ubiquitously expressed in thebody. Despite significant expression in the reproductive system, thereis only limited data about PEDF function in the ovary and uterusreferring mainly to regulation in cancer.

The present inventors have now uncovered a critical role for PEDF in theregulation of hormonal dependent angiogenesis in the reproductive systemand suggest its use in the treatment of a variety of medical conditionsof the female reproductive system, especially those associated withinfertility.

As is illustrated herein below and in FIGS. 1A-D and 2A-B, the presentinventors have found that PEDF is produced in-, and secreted from thegranulosa cells. Furthermore, the present inventors have found thatgonadotropins as well as steroid hormone stimulation regulate PEDF in anopposite manner to the manner they regulate VEGF. This negative feedbackis of much clinical significance as evidenced by animal models ofovarian hyper stimulation syndrome (OHSS) and endometriosis. As shown inFIGS. 4A-D and 5A-D of the Examples section below, the present inventorshave shown that recombinant PEDF is a potent treatment for OHSS in amouse model and further that PEDF/VEGF balance is kept also in theendometrium and is regulated by hormonal changes. All these findingssupport the use of PEDF in the treatment of medical conditions of thefemale reproductive system, especially those associated withinfertility.

Thus, according to an aspect of the invention there is provided a methodof treating or preventing a medical condition selected from the groupconsisting of ovarian hyperstimulation syndrome (OHSS), endometriosis,infertility and polycystic ovarian syndrome in a subject in needthereof. The method comprising administering to the subject apharmaceutical composition comprising an active ingredient consisting ofpigment epithelium-derived factor (PEDF) and a pharmaceuticallyacceptable carrier, thereby treating or preventing the medical conditionselected from the group consisting of ovarian hyperstimulation syndrome,endometriosis, infertility and polycystic ovarian syndrome in thesubject.

As used herein the “subject in need thereof” refers to a mammalianfemale subject (e.g., human) who is diagnosed and optionally treated forany of the above medical conditions. The subject is typically at thereproductive age, however since PEDF improves the quality of the oocytesit can also be used in the treatment in perimenopause. Veterinary usesare also contemplated.

“Pigment epithelium-derived factor (PEDF)” is also known as serpin F1,EPC-1, cell proliferation inducing gene 35 protein and PIG35. Accordingto a specific embodiment, the PEDF protein refers to the human protein,such as provided in the following GenBank Number M76979.

Since the phosphorylated form of PEDF is a potent anti angiogenicfactor, the present invention also contemplates the use of aphosphorylated PEDF (in contrast to non-phosphorylated PEDF) accordingto the present teachings. PEDF is phosphorylated on 3 distinct sitesSer24 and Ser114 by casein kinase 2 (CK2) and on Ser227 by proteinkinase A (PKA). Specifically, contemplated is the use of the triplephosphorylated PEDF. U.S. U.S 20080274967 teaches phosphorylation ofPEDF using CK2 and PKA and is fully incorporated herein.

PEDF can be purified as described in Yanagishe et al. 2006 J.Endocrinol. Metab. 91:2447-2450. Alternatively, PEDF is commerciallyavailable from Biovendor Inc.

Typically, the medical conditions of the female reproductive systemcontemplated herein are associated with infertility (e.g., ovulationdisorders), oftentimes treated with an ovarian stimulating medicationsuch as further described hereinbelow.

Ovarian stimulating medications are the main treatment modalities forwomen who are infertile due to ovulation disorders. These medicationsregulate or induce ovulation. In general, they work like naturalhormones—such as follicle-stimulating hormone (FSH) and luteinizinghormone (LH)—to trigger ovulation. Commonly used fertility drugsinclude:

Clomiphene (Clomid™, Serophene™). This drug is taken orally andstimulates ovulation in women who have polycystic ovary syndrome (PCOS)or other ovulatory disorders. It causes the pituitary gland to releasemore FSH and LH, which stimulate the growth of an ovarian folliclecontaining an egg.

Human menopausal gonadotropin, or hMG, (Repronex). This injectedmedication is for women who don't ovulate on their own due to thefailure of the pituitary gland to stimulate ovulation. Unlikeclomiphene, which stimulates the pituitary gland, hMG and othergonadotropins directly stimulate the ovaries. This drug contains bothFSH and LH.

Follicle-stimulating hormone, or FSH, (Gonal-F, Bravelle™). FSH works bystimulating the ovaries to mature follicles.

Human chorionic gonadotropin, or HCG, (Ovidrel™, Pregnyl™). Used incombination with clomiphene, hMG and FSH, this drug stimulates thefollicle to release its egg (ovulate).

Gonadotropin-releasing hormone (Gn-RH) analogs. This treatment is forwomen with irregular ovulatory cycles or who ovulate prematurely—beforethe lead follicle is mature enough—during hMG treatment. Gn-RH analogsdeliver constant Gn-RH to the pituitary gland, which alters hormoneproduction so that a doctor can induce follicle growth with FSH.

Aromatase inhibitors. This class of medications, which includesletrozole (Femara™) and anastrozole (Arimidex™), is approved fortreatment of advanced breast cancer. Doctors sometimes prescribeletrozole for women who don't ovulate on their own and who haven'tresponded to treatment with clomiphene citrate.

Metformin (Glucophage™). This oral drug is taken to boost ovulation.It's used when insulin resistance is a known or suspected cause ofinfertility. Insulin resistance may play a role in the development ofPCOS.

Bromocriptine (Parlodel). This medication is for women whose ovulationcycles are irregular due to elevated levels of prolactin, the hormonethat stimulates milk production in new mothers. Bromocriptine inhibitsprolactin production.

Since OHSS typically develops in women suffering from POC, endometriosisand/or unexplained infertility subject to induced ovarian stimulation,treatment with PEDF is contemplated for each of these syndromes alone acombination of same. Thus according to a specific embodiment, thesubject may be diagnosed with OHSS, POC and endometriosis.

Specific embodiments of this aspect of the invention are provided infraclassified according to the treated medical condition.

OHSS

Infertility among healthy women is a growing phenomenon (about 10% inthe United States). Ovarian Hyper Stimulation Syndrome (OHSS) is anexaggerated ovarian response to ovulation-induction therapies, thatmight develop after hCG administration. The incidence of OHSS in IVFpatients is estimated at 3-6% for moderate OHSS and 0.1-2% for severeOHSS. Although rare, OHSS can occur in women undergoing ovulationinduction treatment and there have been case reports about spontaneousOHSS in normal cycling women. Therefore the treatment of spontaneousOHSS is also contemplated according to the present teachings. OHSS isassociated with facilitated angiogenesis, which results in vascularhyperpermeability, leakage and shift of fluids into extravascular spacewith consequent clinical manifestations of ascites and hyperviscosity.High Estrogen level serves as a predictor of OHSS. Vascular endothelialgrowth factor (VEGF) is a crucial protein participating in the syndromedevelopment. VEGF is up-regulated in response to elevated Estrogen leveland hCG.

PEDF acts as a strong inhibitor of angiogenesis, known to inhibit theproduction of pro-angiogenic factors, such as VEGF. PEDF isdown-regulated by hCG and high level of Estrogen.

In light of the fact that when OHSS develops, the delicate equilibriumbetween the pro-angiogenic factor VEGF and the anti-angiogenic factorPEDF in the ovary is shattered PEDF treatment can prevent OHSS onset.Hence, administration of PEDF concomitant with gonadotropins stimulationduring the follicular phase is contemplated.

OHSS progression is accompanied by massive and unbalanced angiogenesis.Therefore, treatment of OHSS patients with PEDF during the acute phasewill alleviate the symptoms by restoring the angiogenic balance in theovary.

PCOS

Poly Cystic Ovary Syndrome (PCOS) is the most common endocrine disorderin women of reproductive age. This syndrome may affect 5-10% ofpremenopausal women. The ovaries of PCOS women are characterized byintense-vascularization, which puts them at high risk of developing OHSSas a result of hormonal treatments. Oocytes of PCOS women are often ofpoor quality, leading to lower fertilization, cleavage and implantationrates, and to a higher miscarriage rate. VEGF concentration in the serumand ovaries of PCOS women is elevated, compared to non-PCOS, normalovulating women. Elevated VEGF in follicular fluid of women with PCOS ishighly correlated with immature oocytes, poor fertilization rate anddevelopment of OHSS. Moreover, the elevated circulating VEGFconcentrations in women with PCOS may supply a partial explanation tothe presence of the dense hyperechogenic and highly vascularized stromathat is characteristic of a PCOS. It is the intense vascularization thatmay lead to abnormal growth of the theca interna, which is the site forandrogen steroidogenesis.

PEDF acts as a strong inhibitor of angiogenesis, known to inhibit theproduction of pro-angiogenic factors, such as VEGF.

PEDF administration to PCOS patients undergoing fertility treatment canimprove oocytes quality, fertilization and cleavage rates, number ofembryos, pregnancy rate and decrease the rate of spontaneous abortions.According to some embodiments of the invention, PEDF is administeredbefore or concomitantly with gonadotropins treatment during thefollicular phase. PEDF treatment is also used to overcome the sideeffects of high androgen levels associated with PCOS, such as acne andexcessive hair growth.

Endometriosis

Endometriosis is a pathological condition characterized by ectopicendometrial implants, usually in the peritoneal cavity. Activeendometriosis is characterized by hypervascularization both within, andaround the implant. Endometriosis is manifested during the reproductiveyears; it has been estimated that endometriosis occurs in roughly 5-18%of women. Some symptoms may develop at the site of active endometriosis;the main, but not universal symptom is pelvic pain at variousmanifestations. Other symptoms are: painful sexual intercourse(dyspareunia) or cramping during intercourse, as well as pain duringbowel movements and/or urination. Endometriosis is common in women withinfertility problems. Endometriosis lesions react to hormonalstimulation by proliferation and angiogenesis. There is a high VEGFlevel in the peritoneal fluid of patients with endometriosis and itsproduction is stimulated by both Estrogen and Progesterone.

The present inventors have found that PEDF is expressed in theendometrium and that its expression changes throughout the menstrualcycle; this expression is negatively correlated with VEGF expression.

PEDF level in the endometrium is regulated by gonadotropins and steroidsex hormones. It is suggested that PEDF will decrease VEGF level as wellas vascularization and hence will alleviate endometriosis-relatedsymptoms. Hence, according to an embodiment of the invention symptomaticwomen are treated with PEDF. However, since endometriosis is a chronicsyndrome which affects every day life, a repetitive administration ofPEDF is also contemplated. For example, PEDF may be administered duringferile life span i.e., as long as the ovary secretes the hormonesSuggested treatment is in the luteal phase.

Oocyte Quality

Elevated VEGF level in follicular fluid is associated with poor oocytequality and decreased fertilization and pregnancy rates, especially inolder women and PCOS syndrom.

Administration of PEDF will improve oocytes quality in general, such asin perimenopause.

PEDF can be administered to the subject per se, or in a pharmaceuticalcomposition where it is mixed with suitable carriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofthe active ingredient described herein with other chemical componentssuch as physiologically suitable carriers and excipients. The purpose ofa pharmaceutical composition is to facilitate administration of acompound to an organism.

Herein the term “active ingredient” refers to the PEDF accountable forthe biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous, dermal and transdermaland intramedullary (e.g., using injections or patches).

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuosinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of a disorder or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals (as described in the Examplessection which follows). The data obtained from these in vitro and cellculture assays and animal studies can be used in formulating a range ofdosage for use in human. The dosage may vary depending upon the dosageform employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See e.g.,Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1 p. 1).

Dosage amount and interval may be adjusted individually to providelevels of the active ingredient that are sufficient to induce orsuppress the biological effect (minimal effective concentration, MEC).The MEC will vary for each preparation, but can be estimated from invitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

Exemplary doses are provided infra:

0.02-0.4 mg/kg 0.162-0.32 mg/kg, 0.01-0.2 mg/kg, 0.1-0.4 mg/kg, 0.2-0.4mg/kg or 0.05-0.1 mg/kg.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Thus, the present invention further contemplates a unit dosage formcomprising PEDF.

According to a specific embodiment the unit dosage form comprises 1-500mg PEDF, 1-100 mg PEDF, 1-50 mg PEDF, 1-40 mg PEDF, 1-30 mg PEDF, 1-25mg PEDF, 9-20 mg PEDF, 1-20 mg PEDF or 10-50 mg PEDF.

The unit dosage form can be in the form of an edible unit dosage form(e.g., a tablet); an injectable unit dosage form e.g., an ampoule forinjection, a patch, a pen injector (e.g., prefilled pen cartridges,disposable pen); a unit dosage form for nasal administration (i.e.,nasal spray dose unit).

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as is further detailed above.

As used herein the term “about” refers to ±10%

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

As used herein, the term “preventing” refers to keeping a disease,disorder or condition from occurring in a subject who may be at risk forthe disease, but has not yet been diagnosed as having the disease.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

MATERIALS AND METHODS

Materials

Reagents and Buffers:

Pregnant mare's serum gonadotropin (PMSG; Syncro-part, Sanofi, Paris,France). Human chorionic gonadotropin (hCG), 17-Beta-Estradiol,progesterone and MII medium were purchased from (Sigma, Rehovot,Israel). Dulbecco's modified Eagle's medium (DMEM/Ham F12 1:1, DMEM-F12)and Dulbecco's PBS were acquired from (DPBS; Biological Industries,Beit-Ha'emek, Israel), fetal calf serum (FCS, Invitrogen, Grand Island,N.Y., USA).

The following antibodies and primers were used:

Primary antibodies: anti-CD34 (CEDARLANE Laboratories, NC), anti-VEGF(abeam, Cambridge UK) anti-PEDF (Santa Cruz Biotechnology, CA),anti-actin (Millipore, Temecula Calif.).

Secondary antibodies: monoclonal Cy3-conjugated, mono and poly clonalHRP-conjugated antibodies (Jackson Immunoresearch, PA, USA). RAT AlexaFluor 555 conjugated and rabbit Alexa Flour 488 conjugated (Cellsignaling technology, MA). DNA were stain using Hoechst 3342 (Sigma,Rehovot, Israel) as a nuclear marker.

TABLE 1 Primers, For PCR  Forward 5′CATCCGTA (SEQ ID NO: 1) mouse ActinAAGACCTCTATGCCAAC3  Reverse 5′CAAAGAAA (SEQ ID NO: 2) GGGTGTAAAACGCAGC3′Forward 5′GTGAAGGT (SEQ ID NO: 3) mouse/ GAPDH CGGTGTGAACGG3′ ratReverse 5′GTGATGGC (SEQ ID NO: 4) ATGGACTGTGGTC3′ Forward 5′CATTCACC(SEQ ID NO: 5) rat PEDF GGGCTCTCTACTA3′ Reverse 5′TCAGGGGC(SEQ ID NO: 6) AGGAAGAAGATGAT3 Forward 5′TCTCCTTG (SEQ ID NO: 7) mousePEDF GCGTGGCTTACTTCAA3′ Reverse 5′TGCAGAGA (SEQ ID NO: 8)CTTGGTAAGTTCGCCT3′ Forward 5′AATTGAGA (SEQ ID NO: 9) mouse/ VEGFCCCTGGTGGACA3′ rat Reverse 5′TGAGGTTT (SEQ ID NO: 10) GATCCGCATGATC3′

TABLE 2 Primers, For qPCR Forward 5′CTCATGGAC (SEQ ID mouse/ HPRT1TGATTATGGACAGGA3′ NO: 11) rat Reverse 5′GCAGGTCAG (SEQ IDCAAAGAACTTATAGCC3′ NO: 12) Forward 5′TTCACCCGG (SEQ ID rat PEDFAGCAGTGAT3′ NO: 13) Reverse 5′GCCTCCAGA (SEQ ID ATTGTGTTTGAG3′ NO: 14)Forward 5′CCAAGTCTC (SEQ ID mouse PEDF TGCAGGACATGAAG3′ NO: 15)Reverse 5′GGTTTGCCA (SEQ ID GTAATCTTGCTG3′ NO: 16) Forward 5′CTATGCAGA(SEQ ID rat VEGF TCATGCGGATCA3′ NO: 17) Reverse 5′TATGCTGCA (SEQ IDGGAAGCTCATCTC3′ NO: 18) forward 5′AGGCTGCTG (SEQ ID mouse VEGFTAACGATGAAGC3′ NO: 19) Reverse 5′AGGTTTGAT (SEQ ID CCGCATGATCTG3′NO: 20)

Mouse Model—ICR female mice (5-8 weeks-old; Harlan Laboratories,Jerusalem, Israel) were housed in air conditioned, light controlledanimal facilities of the Sackler faculty of medicine. Animal care was inaccordance with institutional guidelines and was approved by the localauthorities.

Germinal Vesicle (GV) Oocytes—were isolated from ovaries of untreatedmice into MII medium [en-Yosef, D., et al., Tyrosyl-phosphorylatedproteins are involved in regulation of meiosis in the rat egg. MolReprod Dev, 1998. 49(2): p. 176-85] supplemented with 1 μM milrinone(Sigma Rehovot, Israel) to prevent resumption of meiosis and maintainoocytes at the GV stage [Barretto, L. S., et al., Role of roscovitineand IBMX on kinetics of nuclear and cytoplasmic maturation of bovineoocytes in vitro. Anim Reprod Sci, 2007. 99(1-2): p. 202-7].

Oocytes Arrested at Metaphase of the Second Meiotic Division (Mu)—Femalemice were injected with 7 IU of hCG, 48 hours after administration of 5IU PMSG. Ovulated, cumulus enclosed oocytes were taken from theoviductal ampullae into MI medium, 16-18 hours after hCG administration.Cumulus cells were removed by a brief exposure to 400 IU/mlhyaluronidase (Sigma, Rehovot, Israel).

Mouse OHSS Model—The mouse OHSS model was established as described at[Fainaru, O., M. D. Hornstein, and J. Folkman, Doxycycline inhibitsvascular leakage and prevents ovarian hyperstimulation syndrome in amurine model. Fertil Steril, 2009. 92(5): p. 1701-5]. Briefly, PMSG (20IU/day, intraperitoneally (IP)) was administered for 3 days to a 5 weeksold female mice and on forth day, hCG (7 IU, IP) was injected to induceovulation. The control group was treated with single treatment of PMSG(5 IU) and hCG (7 IU) after 48 hours. Vascular permeability wasquantified using the modified Miles vascular permeability assay [Miles,A. A. and E. M. Miles, Vascular reactions to histamine,histamine-liberator and leukotaxine in the skin of guinea-pigs. JPhysiol, 1952. 118(2): p. 228-57], using Evan's blue dye (Sigma, RehovotIsrael) that binds to plasma proteins and leaks with them at sites ofvessel permeability. The abdomen fluid was measured using ELISA readerOD₆₂₀.

Immunofluorescence—GV and MII oocytes were examined. ZP was removed by abrief exposure to alpha-chymotrypsin (50 μg/ml in 1 mM HCl; Sigma, StLouis, Mo.). Oocytes were then fixed by 3% paraformaldehyde (Merck,Gibbstown, N.J.) in DPBS, and washed in a solution of 3% FCS in DPBS(blocking solution). Permeabilization was performed by 10 minutesexposure to 0.05% Nonidet P-40 (NP-40; Sigma, Rehovot, Israel) inblocking solution. Permeabilized oocytes were further incubated for 1.5hours in the presence of the primary PEDF antibody, washed three timesin working solution and incubated for 1 hour with Cy3-conjougatedantibody. Photographs were taken, deconvolved, and processed using aLeica laser confocal microscope (Wetzalr, Germany).

Immunohistochemistry—Paraffin-embedded ovarian sections of 5 weeks weredeparaffinized and subjected to antigen retrieval by microwave treatment(H-3300; Vector Laboratories, Inc, Burlingame). The sections were cooledon ice to room temperature, rinsed in PBS and incubated for 1 hour withPBSTg (0.2% tween). Subsequently sections were blocked with 2% normalhorse serum and incubation overnight with anti-CD34 antibody. Forstaining with anti-PEDF, sections were rinsed in PBS and incubated for 1hour with PBSTg (0.2% tween, 0.2% gelatin). After PBS wash, sectionswere blocked by 10 min incubation in blocking solution (927B; cellmarque) and antibody was apply for overnight incubation.

In the following day sections were washed in PBSTg, and PBS before andafter applying the appropriate secondary antibodies, sections wererinsed and mounted with moviol (Sigma, Rehovot Israel). Photographs weretaken, deconvolved, and processed using using a Leica laser confocalmicroscope.

Cell Culture

Primary Mouse Granulosa Cells—were isolated from estradiol-primed 27days old mice. The ovaries were incubated in hypertonic sucrose/EGTAmedium and transfer into DMEM-F12 in the presence of indomethacin (10μM) and follicles were needle-pricked to release granulosa cells.Isolated cells were plated onto serum-precoated 24-multiwell plates (1ovary per well; Nunc, Copenhagen, Denmark) containing 0.5 ml of DMEM-F12medium [Orly, J., et al., Effects of hormones and protein kinaseinhibitors on expression of steroidogenic enzyme promoters inelectroporated primary rat granulosa cells. Biol Reprod, 1996. 54(1): p.208-18].

Primary Human Granulosa Cells—were obtained from women, aged 22-38years, undergoing IVF treatment in Assaf Harofeh Medical Centre, as ofmale factor infertility. Patients were treated according to the longprotocol guidelines, i.e. received a GnRH agonist at the mid-lutealphase, followed by Follicle Stimulating Hormone (FSH) or humanmenopausal gonadotropin and eventually by the administration of hCG.Granulosa cells were isolated from aspirated follicular fluid afteroocyte retrieval. The follicular fluid was centrifuged at 300 g for 5min at room temperature. The resulting pellets were re-suspended in 10mM Tris, 0.84% NH4 Cl, pH 7.4, to lyse blood cells (15 min shaking at37° C.). Several washings in DPBS eliminated debris. Cells were platedin DMEM-F12, supplemented with penicillin (100 IU/ml, Industries,Beit-Ha'emek, Israel), streptomycin (100 mg/ml, Industries,Beit-Ha'emek, Israel) and 10% FCS. Both primary cultures were incubatedfor an additional 7 days in medium containing 10% FCS. Before harvestingthe cell were serum-starved (0.1% FCS) for 16 hours.

Protein Precipitation—After cells starvation the medium was collectedwith an equal volume of 10% Trichloroacetic acid (TCA, Sigma, St LouisMo.) and incubate the mixture for 16 hours in −20πC. The pellet was thenwashed with ice cold acetone and re-suspend with SDS PAGE loadingbuffer.

Immunoblotting—Cells were grown to subconfluency and then serum starved(0.1% FCS) for 16 hours. After incubation with indicated treatments,cells were lysed in ice-cold radioimmunoprecipitation assay (RIPA, 20 mMTris-HCl pH=7.4, 137 mM NaCl, 10% glycerol, 2% NP-40 or 1% triton X-100,0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA pH=8) buffer and extractswere obtained by centrifugation at 15,000×g at 4° C. Precipitatedprotein was extracted as described above. Aliquots of cellular extractsor precipitated protein were subjected to SDS-PAGE and immunoblottedwith the primary antibodies (PEDF, VEGF, and Actin) followed byincubation with the corresponding horseradish peroxidase-conjugatedsecondary antibody, and developed using Pierce ECL Western BlottingSubstrate (Thermo SCIENTIFIC, IL USA).

PEDF Production—Recombinant PEDF was expressed in E. coli BL21.Bacterial cells and grown at 30° C. to OD_(600 nm)=0.5 to 0.6, inducedby 0.5 mmol/L isopropyl-L-thio-β-D-galactopyranoside for 4 to 5 hours.Pelleted bacterial cells were lysed, and purification of recombinantproteins was performed using ion metal affinity chromatography withNi-NTA His-Bind resin (Merck KGaA, Darmstadt, Germany) according to themanufacturer's protocol. Elution fractions were resolved on SDS-PAGEfollowed by GelCode (Blue Stain Reagent, Thermo SCIENTIFIC, IL USA) orWestern blotting using specific anti-PEDF antibody. Eluates thatexhibited >90% purity were dialyzed against PBS [Konson, A., S. Pradeep,and R. Seger, Phosphomimetic mutants of pigment epithelium-derivedfactor with enhanced antiangiogenic activity as potent anticanceragents. Cancer Res. 70(15): p. 6247-57].

RNA Isolation, Reverse Transcription (RT), PCR and Real-Time PolymeraseChain Reaction (QPCR)—Total RNA was isolated from tissue or cells usinga Trizol reagent according manufacturer instructions (Invitrogen, GrandIsland, N.Y., USA), and quantified with the Nano-Drop spectrophotometer(ND-1000; Thermo scientific, MA, USA). First-strand cDNA was created byRT (Maxima™ Reverse transcriptase, Fermentas UAB) from total 1 μg RNAusing oligo-dt primers. Alternatively, cDNA was retrieved from 100oocytes using high sensitive kit (BioRad Reverse Transcription System,Hercules, Calif.). DNA amplification was with 1 μl of the RT reactionand 50 pmol gene-specific primers in ready mix mixture (Sigma, Rehovot,Israel). The number of cycles was determined following pre-testing arange of cycles in which the product showed linear expression (X18, X25,X35, X45). The PCR products were separated by electrophoresis in a 1.5%agarose gel and visualized by ethidium bromide staining. For detectingchanges in mRNA expression levels the SYBR green reagent was used (SYBR®Green PCR Master Mix, Carlsbad, Calif., USA) along with 15 ng CDNA andspecific primers. The samples were run on ABI Prism 7900 Sequence PCRmachine (Applied Biosystems Foster City, Calif., USA).

Example 1 PEDF is Expressed in the Ovary

The ovarian expression of PEDF was analyzed to study its role as an antiangiogenic factor in the ovary. Histological sections of ICR mouseovaries were immunostained with an anti PEDF antibody (FIG. 1A). PEDFwas found to be localized in both the oocytes and their surroundinggranulosa cells. PEDF was also found in freshly isolated oocytes at thegerminal vesicle (GV) stage and at metaphase of the second meioticdivision (MII) (FIG. 1B). Given that PEDF is a secreted glycoprotein andsince the communication between the oocyte and its surrounding granulosacells is bidirectional [17], the origin of PEDF biosynthesis was tested.Interestingly, while PEDF mRNA was expressed in granulosa cells (FIG.1C), it was not found within the oocytes (FIG. 1D). Hence, it may bethat granulosa cells are the source of PEDF production. To furtherevaluate that, the ability of mouse primary granulosa cells to expressand secret PEDF in vitro was analyzed. Mice primary granulosa cells wereacquired from 27 days old female mice after 3 days of E2 administrationfollowed by culturing for additional 7 days to reach quiescence. PEDFwas detected both in cell lysate and in the culture media (FIG. 2A). Inview of the fact that granulosa cells are the source for PEDFbiosynthesis and secretion in mouse, the same was analyzed in humangranulosa cells can. Human primary granulosa cells were obtained fromwomen undergoing IVF treatments (Helsinki 167/09) pre-cultured for 7days to reach quiescence. PEDF was found to be abundantly expressed inboth culture media and lysates of primary human granulosa cells (FIG.2B).

To conclude PEDF is ubiquitously expressed in the ovaries and may havean important role in regulation of ovarian angiogenesis.

Example 2 Hormonal Regulation of PEDF Secretion

Given that VEGF is the main pro-angiogenic factor in the ovary [3]), andsince VEGF and PEDF were shown to be regulated in an opposite manner inother organs [18], the present inventors have hypothesized that duringthe menstrual cycle PEDF and VEGF are regulated in an opposite manner,thus allowing the maintenance of coordinated angiogenesis in the ovary.

To explore this hypothesis, the present inventors first collectedprimary mouse granulosa cells before and after ovulation and looked forchanges in mRNA levels of PEDF and VEGF (FIGS. 3A-B). While PEDF washighly expressed in primary granulosa cells, its levels decreased aftertreatment with LH. VEGF, on the other hand, presented an oppositepattern: no expression before LH stimulation in granulosa cells.

PEDF expression and secretion from human primary granulosa cells weretested in various time intervals from patients' exposure to hCG (FIG.3E). Furthermore PEDF production increased with time elapse form hCGadministration.

Taken together, these findings indicate that while granulosa cellsabundantly express PEDF, there is only a low expression of VEGF. AfterLH stimulation, PEDF production reduces concomitantly with an increasein VEGF expression. The present inventors assumed that this tandemchange is of much significance in controlling the ovarian angiogenesis.

The follicular growth is accompanied by gradual production of E2 bygranulosa cells, which is known to up regulate VEGF [19, 20].Furthermore, in contiguity to the LH surge, progesterone picks andremains high to support the CL [21, 22]. Therefore, the presentinventors aimed to explore whether E2 and progesterone can regulate PEDFexpression. Mouse and human primary granulosa cells were treated with E2and P and tracked changes in PEDF accumulation in the culture media.Interestingly, stimulation with E2 induced gradual reduction in PEDFwhere P caused abrupt disappearance of the protein (FIG. 3F-Grespectively). These results may imply of sequential regulation ofvascularization.

Example 3 PEDF as a Potential Treatment for Ovarian HyperstimulationSyndrome

It is well established that OHSS is associated with increased VEGFlevels which results in vascular hyperpermeability, leakage and shift offluids into extravascular space [6, 23]. The present results indicatethat there is a negative correlation between PEDF and VEGF, implyingthat in OHSS this balance is disturbed. To test this hypothesis, an OHSSmodel in mouse was established as described by Fainaru et. al [24] andtracked changes of PEDF and VEGF mRNA in ovarian lysates (FIGS. 4A-B).In mice that developed OHSS VEGF mRNA levels increased as compared tocontrol mice [6], while PEDF levels reduced dramatically.

To verify the role of granulosa cells as the main regulators of theovarian vasculature the production of VEGF and PEDF mRNAs were comparedin two granulose cell populations: those isolated from OHSS model miceand those isolated from control mice (Single administration of 5 IU PMSGand 7 IU hCG, standard superovulation). Concurring with our previousresults, granulosa cells from control mice produced smaller amounts ofVEGF mRNA than those from OHSS model mice (3 days of PMSG 20 IUstimulation+single administration of 7 IU hCG). Administration of hCGcaused a slight increase in VEGF mRNA in control granulosa cells but asignificant up-regulation of VEGF mRNA levels in OHSS granulosa cells(FIG. 4C). On the other hand, PEDF mRNA production was down-regulated byincreasing the amount of PMSG while hCG administration had no additiveeffect (FIG. 4D). These results imply that the excess amount of PMSGreflects an imbalance between the production of the main ovarianpro-angiogenic factor, namely VEGF, and the concomitant dramaticdown-regulation of the anti-angiogenic factor, PEDF.

Since it was found that in OHSS, the levels of VEGF are increasedconcomitantly with decreased levels of PEDF, the ability of PEDF toserve as a potential treatment for OHSS was studied. To test thishypothesis the present inventors injected PEDF in parallel to theexcessive administration of gonadotropins, on day 1 and day 3 of PMSGadministration whereas injection of PBS served as a control. Changes inweight and peritoneal vascular leakage (quantified using the Milesvascular permeability assay [25]) were monitored. Treatment with highlevels of gonadotropins led to two-fold increase in body weight comparedto mice treated with regular gonadotropins stimulation (FIG. 5A). PEDFtreatment significantly decreased the generalized edema and weight gainwhich characterizes OHSS.

Importantly, control mice, which were treated with PEDF alone in asimilar timetable, did not lose weight (Data not shown). The presentinventors then tested whether PEDF decreases peritoneal vascular leakageas well (FIG. 5B). High gonadotropin treatment led to an approximate twofold increase in protein leakage into the abdominal cavity as comparedto regular gonadotropins stimulation. PEDF treatment in parallel to thegonadotropin stimulation significantly decreased this leakage (FIG. 5B).Hence, it is possible to conclude that PEDF could serve as a potentialtherapy for OHSS in a murine model by inhibition of vascular leakage.

OHSS development is characterized by rapid and massive angiogenesis.Therefore, the present inventors examined whether PEDF affectsangiogenesis in ovary of OHSS mice model. Ovarian cross sections takenfrom control, OHSS, and OHSS+PEDF treated mice were stained with CD34antibody. OHSS mice model exhibited an exaggerated vascularity ascompared to control mice (FIG. 5C). Concurrent with the present results,the inventors found that PEDF treatment significantly down-regulated theexaggerated ovarian angiogenesis in OHSS mice model (FIG. 5C).

Finally, the present inventors hypothesized that PEDF-induced inhibitionof the overstated angiogenesis is achieved through a direct effect ofPEDF on VEGF. In order to examine this hypothesis, mRNA levels of VEGFin OHSS ovaries were analyzed with or without PEDF treatment andcompared it to the control group. As described for other systems, PEDFadministration reduced VEGF mRNA levels ([18], FIG. 5D).

In view of the above results, it is possible to conclude that treatmentwith PEDF prevents rapid and over-exaggerated angiogenesis, at least inpart, by inhibiting VEGF mRNA production.

Example 4 PEDF in the Endometrium

The above-described results have established a significant role for PEDFas a regulator of angiogenesis in the ovary. Similar to the ovary, theuterine endometrium undergoes cyclic angiogenic changes along themenstrual cycle regulated by pro-angiogenic factors as VEGF [17, 26].Moreover, it was shown that VEGF is the main regulator of endometriumproliferation and migration and it is regulated by E2 and P [27].However, as for the ovary, the anti-angiogenic factor is ill defined.

The present inventors aimed to evaluate whether PEDF is expressed in theendometrium and whether it is regulated in a cyclic manner as well.Seven weeks ICR female mice were synchronized by administration of 5 IUPMSG followed by 7 IU hCG 48 hr later. After synchronization, 2 animalswere sacrificed each day and changes in PEDF and VEGF mRNA along theirnext estrous cycle were monitored (FIG. 6A). PEDF was found to beexpressed in the endometrium and its expression changed throughout thecycle (FIG. 6B). Interestingly, as in the ovary, PEDF expression isnegatively correlated to VEGF (FIG. 6C), suggesting that PEDF mightcounter-regulate VEGF activity in the endometrium as well.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

REFERENCES Other References are Cited Throughout the Application

-   1. Reynolds, L. P., S. D. Killilea, and D. A. Redmer, Angiogenesis    in the female reproductive system. FASEB J, 1992. 6(3): p. 886-92.-   2. Moor, R. M. and R. F. Seamark, Cell signaling, permeability, and    microvasculatory changes during antral follicle development in    mammals. J Dairy Sci, 1986. 69(3): p. 927-43.-   3. Fraser, H. M., Regulation of the ovarian follicular vasculature.    Reprod Biol Endocrinol, 2006. 4: p. 18.-   4. Redmer, D. A. and L. P. Reynolds, Angiogenesis in the ovary. Rev    Reprod, 1996. 1(3): p. 182-92.-   5. Robinson, R. S., et al., Angiogenesis and vascular function in    the ovary. Reproduction, 2009. 138(6): p. 869-81.-   6. Soares, S. R., et al., Targeting the vascular endothelial growth    factor system to prevent ovarian hyperstimulation syndrome. Hum    Reprod Update, 2008. 14(4): p. 321-33.-   7. Chen, S. U., et al., Signal mechanisms of vascular endothelial    growth factor and interleukin-8 in ovarian hyperstimulation    syndrome: dopamine targets their common pathways. Hum Reprod.    25(3): p. 757-67.-   8. Lam, P. M. and C. Haines, Vascular endothelial growth factor    plays more than an angiogenic role in the female reproductive    system. Fertil Steril, 2005. 84(6): p. 1775-8.-   9. Becerra, S. P., et al., Pigment epithelium-derived factor behaves    like a noninhibitory serpin. Neurotrophic activity does not require    the serpin reactive loop. J Biol Chem, 1995. 270(43): p. 25992-9.-   10. Dawson, D. W., et al., Pigment epithelium-derived factor: a    potent inhibitor of angiogenesis. Science, 1999. 285(5425): p.    245-8.-   11. Stellmach, V., et al., Prevention of ischemia-induced    retinopathy by the natural ocular antiangiogenic agent pigment    epithelium-derived factor. Proc Natl Acad Sci USA, 2001. 98(5): p.    2593-7.-   12. Tombran-Tink, J., et al., Organization, evolutionary    conservation, expression and unusual Alu density of the human gene    for pigment epithelium-derived factor, a unique neurotrophic serpin.    Mol Vis, 1996. 2: p. 11.-   13. Petersen, S. V., Z. Valnickova, and J. J. Enghild,    Pigment-epithelium-derived factor (PEDF) occurs at a physiologically    relevant concentration in human blood: purification and    characterization. Biochem J, 2003. 374(Pt 1): p. 199-206.-   14. Cheung, L. W., et al., Pigment epithelium-derived factor is    estrogen sensitive and inhibits the growth of human ovarian cancer    and ovarian surface epithelial cells. Endocrinology, 2006.    147(9): p. 4179-91.-   15. Pollina, E. A., et al., Regulating the angiogenic balance in    tissues. Cell Cycle, 2008. 7(13): p. 2056-70.-   16. Palmieri, D., J. M. Watson, and C. A. Rinehart, Age-related    expression of PEDF/EPC-1 in human endometrial stromal fibroblasts:    implications for interactive senescence. Exp Cell Res, 1999.    247(1): p. 142-7.-   17. Gilchrist, R. B., M. Lane, and J. G. Thompson, Oocyte-secreted    factors: regulators of cumulus cell function and oocyte quality. Hum    Reprod Update, 2008. 14(2): p. 159-77.-   18. Cai, J., et al., Pigment epithelium-derived factor inhibits    angiogenesis via regulated intracellular proteolysis of vascular    endothelial growth factor receptor 1. J Biol Chem, 2006. 281(6): p.    3604-13.-   19. Shimizu, T., et al., Differential effect of follicle-stimulating    hormone and estradiol on expressions of vascular endothelial growth    factor (VEGF) 120, VEGF164 and their receptors in bovine granulosa    cells. J Reprod Dev, 2007. 53(1): p. 105-12.-   20. Danforth, D. R., et al., Vascular endothelial growth factor    stimulates preantral follicle growth in the rat ovary. Biol    Reprod, 2003. 68(5): p. 1736-41.-   21. Stouffer, R. L., Progesterone as a mediator of gonadotrophin    action in the corpus luteum: beyond steroidogenesis. Hum Reprod    Update, 2003. 9(2): p. 99-117.-   22. Shimizu, T. and A. Miyamoto, Progesterone induces the expression    of vascular endothelial growth factor (VEGF) 120 and Flk-1, its    receptor, in bovine granulosa cells. Anim Reprod Sci, 2007.    102(3-4): p. 228-37.-   23. Abramov, Y., et al., Vascular endothelial growth factor plasma    levels correlate to the clinical picture in severe ovarian    hyperstimulation syndrome. Fertil Steril, 1997. 67(2): p. 261-5.-   24. Fainaru, O., M. D. Hornstein, and J. Folkman, Doxycycline    inhibits vascular leakage and prevents ovarian hyperstimulation    syndrome in a murine model. Fertil Steril, 2009. 92(5): p. 1701-5.-   25. Miles, A. A. and E. M. Miles, Vascular reactions to histamine,    histamine-liberator and leukotaxine in the skin of guinea-pigs. J    Physiol, 1952. 118(2): p. 228-57.-   26. Ablonczy, Z., et al., Pigment epithelium-derived factor    maintains retinal pigment epithelium function by inhibiting vascular    endothelial growth factor-R2 signaling through gamma-secretase. J    Biol Chem, 2009. 284(44): p. 30177-86.-   27. Chennazhi, K. P. and N. R. Nayak, Regulation of angiogenesis in    the primate endometrium: vascular endothelial growth factor. Semin    Reprod Med, 2009. 27(1): p. 80-9.

What is claimed is:
 1. A method of treating infertility associated withendometriosis in a subject in need thereof, the method consisting ofadministering to the subject a therapeutically effective amount ofpigment epithelium-derived factor (PEDF) and an ovarian stimulatingmedication, wherein said PEDF is administered subcutaneously,intramuscularly or orally, thereby treating infertility associated withendometriosis in the subject.
 2. The method of claim 1, wherein saidovarian stimulating medication comprises gonadotropin stimulation. 3.The method of claim 1, wherein said administering said therapeuticallyeffective amount of PEDF is effected at a dosage range of 0.02-0.4mg/kg.
 4. The method of claim 1, wherein said administering saidtherapeutically effective amount of PEDF is effected at a dosage rangeof 0.162-0.32 mg/kg.
 5. The method of claim 1, wherein said PEDF isadministered prior to said ovarian stimulating medication.
 6. A methodof treating infertility associated with endometriosis in a subject inneed thereof so as to result in a pregnancy, the method consisting ofadministering to the subject a therapeutically effective amount ofpigment epithelium-derived factor (PEDF) and an ovarian stimulatingmedication, thereby treating infertility associated with endometriosisin the subject.
 7. The method of claim 6, wherein said ovarianstimulating medication comprises gonadotropin stimulation.
 8. The methodof claim 6, wherein said administering said therapeutically effectiveamount of PEDF is effected at a dosage range of 0.02-0.4 mg/kg.
 9. Themethod of claim 6, wherein said administering said therapeuticallyeffective amount of PEDF is effected at a dosage range of 0.162-0.32mg/kg.
 10. The method of claim 6, wherein said PEDF is administeredprior to said ovarian stimulating medication.